Fibronectin binding protein as well as its preparation

ABSTRACT

The present invention relates to a new recombinant hybrid-DNA-molecule comprising nucleotide sequence from S. aureus coding for a protein, or polypeptide, having fibronectin binding properties.

DESCRIPTION

This application is a divisional, of application Ser. No. 08/007,817,filed Jan. 22, 1993, now U.S. Pat. No. 5,320,951, which is acontinuation of application Ser. No. 07/201,028, filed Jun. 1, 1988, nowabandoned.

TECHNICAL FIELD

The present invention relates to a fibronectin binding protein as wellas hybrid-DNA-molecules, e.g. plasmids or phages comprising a nucleotidesequence coding for said protein. Further the invention relates tomicroorganisms comprising said molecules and their use producing saidprotein, as well as the synthetic preparation of said protein.

The object of the present invention is to obtain a minimal fibronectinbinding protein.

A further object is to obtain said protein by means of a geneticengineering technique by using e.g. a plasmid comprising a nucleotidesequence coding for said protein.

A further object is to obtain a possibility of preparing said protein bychemical synthesis.

Further objects will be apparent from the following description.

BACKGROUND OF THE INVENTION

WO-A1-85/05553 discloses bacterial cell surface proteins havingfibronectin, fibrinogen, collagen, and/or laminin binding ability.Thereby it is shown that different bacteria have an ability to bind tofibronectin, fibrinogen, collagen, and/or laminin. It is further shownthat fibronectin binding protein has a molecular weight of 165 kD and/or87 kD, whereby it is probable that the smaller protein is a part of thelarger one.

Fibronectin is a large glycoprotein (M_(r) ca 450 kd) with two similarsubunits, which may vary in moleclar size depending on a complexsplicing pattern of a precursor mRNA (1). The major function offibronectin, which is found in body fluids, blood clots andextracellular matrices, seems to be related to the ability of theprotein to mediate substrate adhesion of most eukaryotic cells (2, 3, 4,5.)

In the late seventies, Kuusela found that fibronectin not only interactswith eucaryotic cells but also binds to cells of Staphylococcus aureus(6). Since this observation, a number of pathogenic microorganisms havebeen shown to bind to fibronectin with a high degree of specificity anda high (7). Fibronectin in the extracellular matrix appears to serve asa substratum also for the adhesion of different microorganism Thebinding of fibronectin may for some microorganisms represent a crucialstep in the colonization of host tissue and development of infection.

Several different cell surface components have been implicated asfibronectin receptors on Gram-positive bacteria including lipotechiocacid (8, 9) and protein (10). In previous studies a fibronectin bindingprotein with a M_(r) of 197-210 kD has been isolated from S. aureusstrain Newman (11, 12) and tentatively identified as a fibronectinreceptor. To further characterize this fibronectin binding protein fromS aureus, the gene for this protein has been cloned in E. coli. Thefibronectin binding domain with in this protein has also been localizedand the behaviour of a fusin protein containing this domain andIgG-binding regions of protein A will be disclosed below.

DESCRIPTION OF THE INVENTION

It has now surprisingly been found possible to obtain ahybride-DNA-molecule comprising a nucleotide sequence coding for aprotein or a polypeptide having fibronectin binding properties. Asevident from below the following nucleotide sequence [SEQ ID NO. 4] ispresent in the gene coding for said protein: ##STR1##

The invention further comprises a plasmid or phage comprising anucleotide sequence coding for said fibronectin binding protein.

The invention further comprises a microorganism comprising at least onehybrid-DNA-molecule according to the above.

The invention further comprises a method for producing a fibronectinbinding protein whereby at least one hybrid-DNA-molecule of above isintroduced into a microorganism, cultivating said microorganism in agrowth medium, and isolating the protein thus formed by means of anaffinity chromatography on a fibronectin bound to an insolubilizedcarrier followed by ion exchange chromatography.

A further aspect of the invention comprises a chemical synthesis of thefibronectin binding protein, whereby an amino acid sequence is built upbased on said nucleotide sequence encoding for said protein startingfrom the C-terminal histidine which is stepwise reacted with theappropriate amino acid, whereby it is finally reacted with glycine atthe N-terminal end, to form the fibronectin binding peptide region.

Appropriate carrier proteins can be coupled to the amino acid sequenceas well, such as IgG binding regions of protein A.

The invention will be described in the following with reference to theexamples given, however, without being restricted thereto.

EXAMPLE 1 Screening of a Gene Bank For Fibronectin Binding Protein(FNBP)

A gene bank in plasmid pBR322 of chromosomal DNA from Staphylococcusaureus strain 8325-4 earlier described by (13) was screened for clonesexpressing FNBP. E. coli clones were lysed and the lysates were testeofor their ability to inhibit the

of ¹²⁵ I-fibronectin to cells of S. aureus Cowan I as described in themethod section. To simplify screening the clones were pooled in lots of25, lysed and tested. Out of the 22 pools tested, the one with thehighest inhibitory activity was retested in 5 pools of 5. Finally, theindividual clones in one positive pool were tested resulting in theisolation of one single positive clone. The plasmid in the positiveclone was called pFR001.

This plasmid pFR001 in an E. coli strain 259 has been deposited at theDeutsche Sammlung von Mikroorganismen (DSM) and has thereby beenallocated the deposition number 4124.

Isolation of the Staphylococcal Fnbp From E. Coli pFR001

The E.coli clone, positive for FNBP, was grown in LB-medium at 37° C. tothe stationary growth phase. The bacterial cells were centrifuged andlysed by an osmotic shock procedure (14). To exclude that inhibitoryeffect of the shock lysate on the binding of ¹²⁵ I-fibronectin to S.aureus cells is due to proteolytic activity,lysate was added to theincubation mixture and the amount of ¹²⁵ I-fibronectin bound to S.aureus was determined after 1,2,3, and 4 hrs of incubation. Throughoutthis incubation period, the level of inhibition caused by the lysateremained constant (e.g. 50%) suggesting that the observed inhibition wasnot due to a progressive degradation of the ¹²⁵ I-labelled ligand orcorresponding receptor.

If the inhibitory activity is due to the presence of FNBP-likestructures in the lysate, these should express a specific affinity forfibronectin. The lysate, was therefore analysed by affinitychromatography on a column of fibronectin-Sepharose as described inMaterials and Methods (FIG. 1A). The purification was about 30 fold.Further fractionation was achieved by subjecting the affinity purifiedmaterial to ion-exchange chromatography using a mono Q column fitted onto a FPLC-system. In this fractionation step two major peaks wereobtained (FIG. 1B). Analyses by polyacrylamide gel electrophoresisindicated th at the two peaks contained proteins of molecular weights165 and 87 kD, respectively (FIG. 2). Both components inhibited thebinding of ¹²⁵ I-fibronectin to S. aureus. The fraction containing the165 kD protein had a specific inhibitory activity of 220 units/qgrepresenting a 430-fold purification from the original shock lysate andit was 30 times more active than the 87 kD protein on a molar basis(data not shown).

Amino acid analysis of the two proteins showed that they had a verysimilar amino acid composition which also closely resembled thatdetermined for the native FNBP isolated from S. aureus strain Newman(Table 1). The immuological relationship of 165 kD protein isolated fromE.coli pFR001 to the native FNBP isolated from S. aureus strain Newmanwas analyzed. The 165 kD protein was ¹²⁵ I-labelled andimmunoprecipitaited with increasing dilutions of an antibody to S.aureus strain Newman. A 300-fold dilution of the antiserum precipitatd50% of the ¹²⁵ I-labelled protein. Unlabelled 165 and 87 kD proteinswell as the native FNBP interferred with the immunoprecipitation of thelabelled 165 kD protein (data not shown). These observations suggestthat the 165 and 87 kD proteins isolated from S. aureus strain Newmanare closely related to fibronectin binding protein (FNBP).

Identification of The Region of The Fnbp-Gene Coding for The BindingActivity

The size of the insert in plasmid pFR001 is about 6.5 kbp. Therestriction map of the insert is shown in FIG. 3(A). In order todetermine the transcription direction and to localize the region codingfor the binding function, the PstI fragment of about 3.7 kbp wasrecloned in the PstI site of pBR322. This results in loss of theampicillin resistance phenotype conferred by this plasmid. Eight suchclones were obtained, five of which were positive and three negative forfibronectin binding activity. One of each was tested for the orientationof the PstI fragment. The plasmid of the positive clone pFR004 had theEcoRI site closest to the Amp-promoter and that of the negative clonehad the reverse orientation. These data indicated that the transcriptionorientation is from EcoRI to PstI on the 3.7 kbp EcoRI-PstI fragment andthat at least a part of the fnbp-gene, which codes for the bindingfunction, is located on this fragment. To verify this, the Amp-promoterin pFR004 was removed by EcoRI digestion followed by religation of theplasmid. The resulting plasmid pFR008 does not express fibronectinbinding activity. In pFR001 the endogenous promoter is thus presumablylocated somewhere on the left hand side of the EcoRI site as indicatedby the arrow in FIG. 3A). By a plasmid construction (not described indetail here) the EcoRI (in the insert)-SalI (in pBR322) fragment frompFR001 was introduced into pFR008. The fibronectin binding activity wasthen regained, due to the restoration of the endogenous promoter fromthe fnbp-gene.

Knowing the transcription direction of the fnbp-gene (from left to rightas drawn in FIG. 3(A)) fusions were made to the gene for staphylococcalprotein A. An expression/secretion vector called pRIT3 based on theprotein A gene with restriction enzyme multilinker has been constructed(15). The multi-linker is placed immediately downstream of the lastIgG-binding region, thus eliminating the C-terminal cellwall bindingregion of protein A. The 3.7 kbp EcoRI-PstI fragment from pFR001 wasinserted into this vector expecting it to, encode a protein A-FNBPfusion protein, providing the reading frame was correct. This wasobviously the case since the clone containing this plasmid, calledpFR013, is positive in tests for both protein A and FNBP.

Plasmid pFR013 was treated with exonuclease Bal31 in order to identify aregion smaller than the 3.7 kbp insert coding for the fibronectinbinding activity. The plasmid was cleaved with EcoRI or Pst1 (at the 5'and the 3' end of the coding region, respectively), treated for varioustimes with Bal31 and religated. During ligations a 20-fold excess of anEcoRI linker was added in oder to introduce EcoRI sites at newpositions. FIG. 3(C) shows the deletions obtained from either the 3' orthe 5' end and the corresponding fibronectin binding activity. A regionof the gene of about 700 bp coding for binding activity is locatedbetween the end points of deletions number 56 (deletion from the 5 end)and number 22 (deletion from the 3 end). From deletion plasmid number56, the 900 bp region from the newly introducted EcoRI site to the PvuIIsite was subcloned. This fragment was cloned into pUC18 cleaved withEcoRI and SmaI. The resulting plasmid, which encodes a fusion proteinwith both β-galactosidase and fibronectin binding activity, is calledpFR015. The fragment can be recloned from pFR015 by EcoRI and BamHIcleavages because of the multilinker in pUC18.

Restriction fragments were also subcloned as indicated in FIG. 3(B). Inthe case of EcoRI-PstI and EcoRI-ClaI fragments the protein A expressionvector pRIT3 was used. Fragments BalI-PvuII and BalI-HincII weresubcloned and expressed in pUC18. In all cases, except for theEcoRI-ClaI fragment, fusion proteins with fibronectin binding activitywere obtained. The negative result in the case of the ClaI-ClaI subclonemay be due to the insert appearing in the wrong reading frame.

Production and Characterization of a Fusion Protein, ZZ-FR

The yield of the 165 kD protein (FIG. 2) from an osmotic shock lysate ofE. coli HB101 cells carrying the plasmid pFR001 was approximately 40/μgper liter culture medium. Even if there were losses due to degradationof the high molecular weight compound during the purification all dataindicated that the fnbp-gene with the endogenous promoter is weaklyexpressed in E. coli. In order to improve the level of expression weused a recently developed expression system, which allows heterologousproteins to be secreted to the growth medium of E. coli (16). Theplasmid vector used, pEZZ318, contains two synthetic slightly modifiedIgG-binding domains of the gene for staphylococcal protein A preceededby the promoter and signal sequence of the same gene. A BalI-PvuIIfragment of approximately 600 bp of the fnbp-gene (FIG. 3(B)) cloned inpUC18 was recloned into pEZZ318 cleaved with EcoRI and HindIII. Afterligation and transformation pEZZ-FR was isolated. This plasmid encodes afusion protein consisting of the IgG binding product ZZ and fibronectinbinding region FR of the FNBP.

For production of the ZZ-FR protein E. coli strain HB101 carrying theplasmid pEZZ-FR was grown overnight in Trypticase Soy Broth. Bacteriawere removed by centrifugation and the growth medium was passed throughan IgG-Sepharose Fast Flow column. After washing the column withTST-buffer (50 mM Tris-HCl pH 7.4 150 mM NaCl, 0,05% Tween^(R) 20) theZZ-FR protein was eluted with 0,5 M acetic acid titrated to pH 2.8 usingammonium acetate. Approximately 50 mg protein was eluted from the columnper liter of growth medium applied.

After lyophilization the eluted material was analyzed by SDS-PAGE, whichrevealed a major protein band at approximately 63 kD (FIG. 4). Inaddition, bands corresponding to smaller fragments appeared, probablydue to proteolytic degradation of the fusion protein. The intact proteinA which was run on the same gel appeared as a diffuse band around 56 kD.The proteins in the gel were electrophoretically transferred to anitrocellulose paper and probed with a ¹²⁵ I-labelled 29 kD fibronectinfragment. FIG. 4, lanes C and D, shows that the fusion protein but notintact protein A, binds the radiolabelled fibronectin fragment.

Further evidence for the fibronectin binding ability of the ZZ-FR fusionprotein was obtained by affinity chromatography on a Sepharose columnsubstituted with the 29 kD fibronectin fragment. The fusion protein wasbound to the column and was eluted from the affinity matrix with 6MGuHCl (FIG. 5A). The fibronectin binding activity of the ZZ-FR fusionprotein was apparently located in the FR-region since intact protein Adid not bind to the affinity matrix (FIG. 5B). The portion of the ZZ-FRfusion protein preparation that did not bind to the affinity matrixconsisted of proteins with M_(R) lower than of the intact fusion protein(FIG. 6, lane A) whereas the material binding to the column consisted ofan almost pure preparation of intact 63 kD ZZ-FR fusion protein (FIG. 6,lane B).

In oder to determine how much of the fibronectin binding capacity ofstaphylocaccal cells that can be ascribed to the cloned FNBP, theinhibitory activity of the fusion protein ZZ-FR was quantified. As shownin FIG. 7 the fusion protein totally inhibited the binding of ¹²⁵I-labelled 29 kD fragment as well as intact fibronectin to cells of bothS. aureus strains Newman and 8325-4, protein A, which was used as acontrol, did not inhibit the binding (FIG. 7).

After the report by Kuusela (6) that S. aureus binds to fibronectin muchwork has been focused on attempts to identify the bacterial component(s)responsible for the binding. The rationale for these studies has beenthat binding of pathogenic bacteria to fibronectin may represent amechanism of tissue adherence of crucial importance in the early stagesof an infection. Proteins purified by affinity chromatography onimmobilized fibronectin, have been implicated in the binding ofstaphylococcal cells to fibronectin. However the reported lecularweights of the fibronectin binding proteins vary from 18 kD (10) all theway up to 197 and 210 kD (11, 17). The main reason for the heterogenietyin molecular size may be proteolytic degradation of the proteins duringthe isolation procedures.

In the present disclosure the cloning in E. coli of a gene coding for afibronectin binding protein from S. aureus strain 8325-4 is disclosed.When the fnbp-gene is expressed in E. coli from the endogenous promoterthe protein can be isolated from the periplasm by osmotic shock. Thisindicates that not only the promoter but also the signal peptide isfunctional in E. coli.

Although the proteins coded for by the cloned fnbp-gene have molecularweights of 165 and 87 kD (FIG. 2), which is smaller than the FNBPisolated from S. aureus strain Newman (M_(R) =210 kD) (12), their aminoacid compositions closely resemble that of the native protein (Table 1).Furthermore, antibodies raised against the native FNBP cross-react withthe 165 and 87 kD proteins. These data strongly suggest that thestructure of the proteins coded for by the cloned gene from S. aureusstrain 8325-4 resembles that of native FNBP from S. aureus strainNewman. The 87 kD protein may be the result of pretermination at thetranscriptional or translational level or alternatively proteolyticcleavage of the 165 kD protein. At present it cannot be explained whythe 165 kD protein is as much as 30 times more active than the 87 kDprotein in inhibition of fibronectin binding to S. aureus cells.

By deletion mapping using Bal31-clevage and subcloning of restrictionfragments the domain of the fnbp-gene encoding the fibronectin bindingactivity had been located to a region of approximately 350 bp (FIG.3(B)). A fragment of the gene of approximately 600 bp covering these 350bp was ligated directly to a tandemly repeated sequence (zz) of asynthetic IgG-binding domain of the protein A gene preceeded by theprotein A promoter and signal sequence in expression vector pEZZ318(16). The resulting fusion protein (ZZ-FR), which has a molecular weightof approximately 63 kD as determined by SDS-PAGE (FIG. 4), contains 126amino acids of the ZZ domain followed by approximately 200 amino acidsencoded by the 600 bp insert from the fnbp-gene The C-terminal end ofthe protein consists of amino acids which are the result of an out offrame read-through into the lacZ' gene of the vector until a translationstop codon is reached. The fusion protein (ZZ-FR), which is expressed ata high level and secreted to the growth medium of E. coli, is easilyisolated by affinity chromatogtaphy making use of the IgG-bindingability of the ZZ-domain.

The ZZ-FR protein was bound to the 29 kD NH₂ -terminal domain offibronectin and completely inhibited the binding of intact fibronectinto S. aureus (FIGS. 5 and 6). These data indicate that under theincubation conditions used other proteins, recognizing domains outsidethe 29 kD N-terminus of fibronectin, are not expressed by thestaphylococcal cells. Furthermore the fibronectin binding activity ofthe FNBP has been localized to a fairly small segment of the protein.Recent analysis of the native 210 kD FNBP isolated from S. aureus strainNewman demonstrated that this protein is multivalent and one molecule ofthe FNBP is capable of binding 6-9 fibronectin molecules (12). Thecloned fnbp-gene is derived from strain S. aureus 8325-4. If there areno differences between strains of S. aureus one would except theFR-region to contain several repeating sequences. This question was alsoanswered by sequence analysis of the cloned fnbp-gene. The sequence ofthe FR-region having FNBP-properties is given in FIG. 8

There is reason to believe that the FR binding activity is related tothe each of the three 38 amino acids repeats as the Bal1-PvuII-fragmentencodes for a binding activity (cf. FIG. 3); as the Bal1-HincII-fragmentencodes for a binding activity; and as the HincII-PvuII-fragment doesnot encode for binding activity. Furthermore, the one single 38 aminoacids repeat is functional in binding fibronectin, since a synthezised38-amino acids long peptide (38(2)-repeat) has binding ability, as shownin FIG. 9. Each of the three 38 amino acids repeats are very homologous

EXAMPLE 2

Chemical synthesis of a polypeptide based on the nucleotide sequencecoding for the fibronectin binding domain of 38 amino acids (38(2)repeat) was performed by building up the amino acid sequencecorresponding to said nucleotide sequence starting from the C-terminalhistidin and stepwise reacting with the appropriate amino acid andfinally reacting with the glycine at the N-terminal end, in a solidphase synthesis according to the method by K. B. Merrifield, J. Am.Chem. Soc. 86, pp. 304, (1964). Hereby the polypeptide corresponding tothe second 38 amino acid repeat was synthezised, as well as threefurther polypeptides being parts of this 38-repeat, viz: 1) the polypeptide covering amino acids 1-19, 2) the polypeptide covering aminoacids 9-30, and 3) the polypeptide covering amino acids 20-38, were allsynthezised according to the same method.

The fibronectin binding ability of the complete 38-repeat, as well as ofthe 1-19 amino acid polypeptide, the 9-30 amino acid polypeptide, the20-38 amino acid polypeptide, as well as a mixture of these threesmaller polypeptides was tested, and the result is given in FIG. 9. Thefragments of the complete 38-repeat were synthezised in order to checkif fibronectin binding is dependent upon the complete 38-repeat, asanticipated, or if fibronectin binding properties could be furtherconfined to some smaller regoin of the 38 amino acid sequence. Asevident from FIG. 9 the fibronectin binding property is only present inthe complete 38 amino acids peptide.

Materials and Methods

Bacterial Strains and Plasmids.

A gene bank in pBR322 of chromosomal DNA from Staphylococcus aureusstrain 8325-4, earlier described (13) was screened for clones expressingfibronectin binding activity. E. coli strains HB102, (18) and JM105(19), were used in subcloning and expression experiments. The plasmidvectors used were pBR322 (20) pUC18 (21) and the protein A vectors pRIT3(15) and pEZZ318 (16). S. aureus strains Cowan I, Newman and 8325-4 wereused in the assay of the fibronectin binding protein (FNBP).

Microorganism Growth Medium

At the culture of E. coli bacteria the following medium was used. Theamounts given relates to 1 liter of medium.

    ______________________________________                                        Trypton Soy Broth (Oxoid Ltd,                                                                         30     g                                              Basingstoke, Hants, GB)                                                       Yeast Extract (Oxoid)   10     9                                              D-glucose               40     9                                              NH.sub.4 Cl             2,5    g                                              Na.sub.2 HPO.sub.4.2H.sub.2 O                                                                         7,5    g                                              KH.sub.2 PO.sub.4       3,0    g                                              Na.sub.2 SO.sub.4.10H.sub.2 O                                                                         2,5    g                                              MgSO.sub.4.7H.sub.2 O   0,2    g                                              CaCl.sub.2.2H.sub.2 O   0,5    mg                                             FeCl.sub.3.6H.sub.2 O   16,7   mg                                             ZnSO.sub.4.7H.sub.2 O   0,18   mg                                             CuSO.sub.4.5H.sub.2 O   0,16   mg                                             MnSO.sub.4.4H.sub.2 O   0,15   mg                                             CoCl.sub.2              0,10   mg                                             NaEDTA                  20,1   mg                                             ______________________________________                                    

Assay of Fibronectin Binding Protein (FNBP)

Quantitation of fibronectin binding to cells of S. aureus has beendescribed earlier (10). If not otherwise stated, 10⁹ cells of S. aureusCowan I are incubated with ¹²⁵ I-labelled fibronectin or the 29 kD NH₂-terminal fragment of fibronectin in PBS containing 1 mg/ml bovine serumalbumin in a total volume of 0,3 ml. After incubation for 2 hours at 22°C. the radioactivity bound to the cells is measured in a gamma counter.

Lysates of E. coli clones prepared in Tris-HCl buffer, pH 8,1,containing lysozyme EDTA as earlier described (13), were analysed forfibronectin binding activity by measuring their ability to compete withstaphylococcal cells for binding the ¹²⁵ I-labelled 29 kD NH₂ -terminalfragment of fibronectin. The amount of FNBP able to inhibit binding to50% is considered as one unit of activity.

An osmotic shock procedure was used to release proteins from theperiplasmic space of E. coli (14).

Purification of FNBP

The purification is based on affinity chromatography onfibronectin-Sepharose followed by ion-exchange chromatography.

Human fibronectin was prepared from outdated blood by the method known(22). The fibronectin was then dialysed against 20 mM Tris-HCl, pH 8,3and concentrated on a DEAE-column. The coupling of fibronectin toSepharose SL-4B was done by a bromocyan activation procedure as known(23).

The E. coli lysate was pumped onto the affinity column which wassubsequently washed with 0,5M ammonium acetate until the baseline wasstable (about four column volumes). The FNBP was then eluted with 0,4Macetic acid and either neutralized with ammonia or lyophilized. Afterdialysis against 10 mM ammonium acetate with pH adjusted to 7,6 withammonia a further fractionation step was performed by ion-exchangechromatography on a Pharmacia FPLC equipment using a mono Q column.

Nucleotide Sequence Analysis

The nucleotide sequence was determined in accordance with the methodsdescribed by Maxam, A.M. et al (27).

Amino Acid Analysis

The amino acid composition was determined using a Durrum D-500 analyzer.Samples were hydrolyzed in 6M HCl containing 2 mg/ml phenol for 24 hoursat 110° C. One sample was also oxidized with performic acid in oder todetermine cystein and methionine. Norluecin was added as an internalstandard. Protein was determined according to (24) using bovine serumalbumin as a standard.

Electrophoresis

If not indicated otherwise SDS-polyacrylamide gel electroporesis wasperformed in 5-15% gradient gels. The gels were stained with Coomassiebrilliant blue, de-stained and photographed.

Radioimmunoassay Procedure

The purified FNBP with an estimated molecular weight inSDS-polyacrylamide gel electrophoresis of 165 kD was labelled with ¹²⁵I-odine by the chloramine-T method as known (25). After the iodinationthe material was rechromatographed on a fibronectin-Sepharose column.

The incubation mixture contained ¹²⁵ I-FNBP (3 400 cpm in 10/μl) wasmixed with various dilutions of a rabbit antiserum (containingantibodies directed against S. aureus strain Newman), in incubationbuffer (PBS, 0,1% Triton X-100 and 0,02% sodium azid) in a volume of 0,2ml.

Samples were for 2 hours at 20° C. to allow antigen-antibody reaction.0,1 ml of 10% suspension (w/v) of protein A-Sepharose in PBS was addedand the mixture was incubated for another hour. The incubation wasstopped by adding another 1,7 ml of incubation buffer to the samples.After centrifugation at 2000 rpm for 3 min the supernatants were suckedoff. The pellets were washed twice in incubation buffer and theradioactivity associated with the protein A-Sepharose was measured in agammacounter.

Restriction Endonucleases and Other Enzymes

Restriction enzymes, T4 DNA ligase and Bal31 were purchased from BRL andused according to their recommendations. Other methods involving DNAtechniques were essentially as known (26).

                  TABLE 1                                                         ______________________________________                                        Comparison of amino acid compositions of fibronectin binding                  proteins (87 and 165 kD) isolated from E. coli pFR001 and the                 native FNBP isolated from S. aureus strain Newman (210 kD).                                 Composition (mol %)                                             Amino acid      210 kD.sup.a)                                                                            165 kD  87 kD                                      ______________________________________                                        Aspartic acid/asparagine                                                                      15.4       14.6    13.4                                       Threonine       9.7        10.7    10.3                                       Serine          7.4        6.5     8.0                                        Glutamic acid/glutamine                                                                       17.9       17.1    15.1                                       Proline         6.3        6.2     5.8                                        Glycine         8.9        7.9     8.4                                        Alanine         5.3        4.6     4.7                                        Half-cystine    0.1        0.2     n.d.                                       Valine          7.2        7.8     8.6                                        Methionine      0.6        0.6     n.d.                                       Isoleucine      4.3        4.7     3.8                                        Leucine         4.1        4.0     4.6                                        Tyrosine        2.1        2.3     4.1                                        Phenylalanine   2.4        2.0     3.6                                        Histidine       1.0        3.2     3.0                                        Lysine          5.3        6.3     6.6                                        Tryptophan      n.d.       n.d.    n.d.                                       Arginine        1.9        1.2     --                                         ______________________________________                                         .sup.a) from Froman et al. (1987), (12).                                      n.d. = not determined                                                    

The present fibronectin binding protein can be used for immunization,whereby the protein, preferably in combination with a fusion protein tocreate a large antigen to respond to, is injected in dosages causingimmunological reaction in the host mammal. Thus the fibronectin bindingprotein can be used in vaccination of ruminants against mastitis causedby Staphylococcal infections. The fibronectin binding protein of thisinvention has shown to form antibodies against a staphylococcal mastitisin a mouse model as shown in the Table below.

TABLE

Experimental mouse mastitis produced by S. aureus strain SA 113(83A) ina dose of 1,0×10³ cfu. Evaluation of immunization using 15/μg proteinper mouse of the fibronectin binding protein expressed from E. colicontaining plasmid pFR001 as identified herein.

                                      TABLE                                       __________________________________________________________________________    Experimental mouse mastitis produced by S. aureus strain SA 113(83A) in       dose of 1,0 × 10.sup.3 cfu. Evaluation of immunization using 15/ug      protein per                                                                   mouse of the fibronectin binding protein expressed from E. coli               containing plasmid pFR001 as identified herein.                                     No. of           No. of                                                       mammary                                                                             Gross examination                                                                        mammary                                                                             Microscopic                                      Group of                                                                            glands                                                                              type of lesion (%)                                                                       glands                                                                              type of lesion (%)                               mice  inocul.                                                                             +++ ++ + 0 invest.                                                                             A B C1                                                                              C2                                                                              C3                                       __________________________________________________________________________    Vaccinated                                                                          30     3   8 83                                                                              10                                                                              11     9                                                                              0  0                                                                              91                                                                              0                                        (FNBP)                                                                        Control                                                                             38    50  16 32                                                                               3                                                                               8    38                                                                              0 25                                                                              38                                                                              0                                        __________________________________________________________________________     Mastitis: +++ = gross; ++ = mediumgrade; + = Low grade; 0 = no macroscopi     changes;                                                                      A = consistently nonreactive, total necrosis; B = advanced regressive         changes + slight inflammatory reaction; C1 = disseminated inflammatory        reaction + local necrosis; C2 = disseminated inflammatory reaction; C3 =      local inflammatory reaction; 0 = no reaction.                            

As evident from the Table above a consistent immunization is obtainedusing the FNBP as expressed by the E. coli containing the plasmidpFR001.

Further, the fibronectin binding protein can be used to block aninfection in an open skin wound by wound treatment using the fibronectinbinding protein in a suspension. Thus the fibronectin binding proteincan be used for the treatment of wounds, e.g. for blocking proteinreceptors, or for immunization (vaccination). In the latter case thehost body produces specific antibodies, which can protect againstinvasion of bacterial strains comprising such a fibronectin bindingprotein. Hereby the antibodies block the adherence of the bacterialstrains to damaged tissue.

Examples of colonization and tissue damage are:

a) colonizing of wounds in skin and connective tissue, which wounds havebeen caused by a mechanical trauma, chemical damage, and/or thermicaldamage;

b) colonizing of wounds on mucous membranes, such as in the mouthcavity, or in the mammary glands, urethra, or vagina;

c) colonizing on connective tissue proteins, which have been exposed bya minimal tissue damage (microlesion) in connection with epithelium andendothelium (mastitis, heart valve infection, hip exchange surgery).

When using the present FNBP, or the 38 amino acid polypeptide, for thepurpose of immunization (vaccination) in mammals, including man, theprotein, or polypeptide is dispersed in sterile, isotonic salinesolution, optionally while adding a pharmaceutically acceptabledispersing agent. Different types of adjuvants can further be used inorder to sustain the release in the tissue, and thus expose the proteinor the peptide for a longer time to the immundefense system of a body.

A suitable dosage to obtain immunization is 0,5 to 5/μg of FNBP, orpolypeptide, per kg bodyweight and injection of immunization. In orderto obtain a durable immunization, vaccination should be carried out atmore than one consecutive occasions with an interval of 1 to 3 weeks,preferably at three occasions.

When using the present FNBP, or polypeptide, for topical, localadministration the protein is dispersed in an isotonic saline solutionto a concentration of 25 to 250/μg per ml. The wounds are then treatedwith such an amount only to obtain a complete wetting of the woundsurface. For an average wound thus only a couple of milliliters ofsolution are used in this way. After treatment using the proteinsolution the wounds are suitably washed with isotonic saline or anothersuitable wound treatment solution.

Further the fibronectin binding protein as well as the minimalfibronectin binding site polypeptide., of the present invention can beused to diagnose bacterial infections caused by Staphylococci strains,whereby a fibronectin binding protein of the present invention isimmobilized on a solid carrier, such as small latex or Sepharose^(R)beads, whereupon sera containing antibodies are allowed to pass andreact with the FNBP thus immobilized. The agglutination is then measuredby known methods.

Further, the FNBP, or the polypeptide can be used in an ELISA test(Enzyme Linked Immuno Sorbent Assay; E Engvall, Med. Biol. 55, 193,(1977)). Hereby wells in a polystyrene microtitre plate are coated withthe FNBP, and incubated over night at 4° C. The plates are thenthoroughly washed using PBS containing 0.05% TWEEN 20, and dried. Serialdilution of the patient serum were made in PBS-Tween, were added to thewells, and incubated at 30° C. for 1.5 hrs. After rinsing antihuman-IgGconjugated with an enzyme, or an antibovine-IgG conjugated with anenzyme, respectivel, horseradishperoxidase or an alkaline phosphatase,was added to the wells and incubated at 30° C. for 1,5 hrs, whereuponwhen the IgG has been bound thereto, and after rinsing, an enzymesubstrate is added, a p-nitrophosphate in case of an alkalinephosphatase, or ortophenylene diamine substrate (OPD) in case aperoxidase has been used, respectively. The plates comprising the wellswere thus then rinsed using a citrate buffer containing 0.055% OPD, and0.005% H₂ O₂, and incubated at 30° C. for 10 min. Enzyme reaction wasstopped by adding a 4N solution of H₂ SO₄ to each well. The colourdevelopment was measured using a spectrophotometer.

Depending on the type of enzyme substrate used a fluoroscensemeasurement can be used as well.

Another method to diagnose Staphylococci infections is by using the DNAgene probe method based on the FNBP sequence or the 38 amino acidpolypeptide sequence. Thereby the natural or synthetic DNA sequences areattached to a solid carrier, such as a polystyrene plate as mentionedabove, by e.g. adding a milk in the case of diagnozing a mastitis, tothe surface. The DNA gene probe, optionally labelled enzymatically, orby a radioactive isotope is then added to the solid surface platecomprising the DNA sequence, whereby the DNA gene probe attaches to thesequence where appearing. The enzyme or the radioactive isotope can thenreadily be determined by known methods.

Above the term fibronectin binding protein includes any of the 38 aminoacid polypeptide sequences as well, which 38 amino acid polypeptidesequences forms the minimal fibronectin binding site of the completeprotein.

REFERENCES

8. Beachey, E. H. and Simpson, W. A (1982). Infection 10, 107-110.

20. Bolivar, F., Rodriquez, R. L., Greene, P. J., Betlach, M. C.,Heyneker, H:L., Boyer, H. W., Crosa, J. H. and Falkow, S. (1977). Gene,2, 95-113.

18. Boyer, H. W. and Roulland-Dussoix, D. (1969). J. Mol.Biol: 41,459-472.

9. Courtney, H. S., Ofek,I., Simpson, W. A., Hasty, D. L. and Beachey,E. H. (1986). Infect. Immun. 53, 454-459.

11. Espersen, F. and Clemmensen, I. (1982). Infect. Immun. 37, 526-531.

17. Froman, G., Switalski, L. M., Guss, B., Lindberg., M., Hook, M. andWadstrom, T. (1986) In Lark, D. L. ed. Protein-Carbohydrate Interactionsin Biological Systems. Academic Press, London, pp. 263-268.

12. Froman, G., Switalski, L. M., Speziale, P. and Hook, M. (1987) inpress. J. Biol. Chem.

25. Hunter, W. M. (1978) Radioimmunoassay. In: Wier, K. M. ed. Handbookof Experimental Immunology. London Blackwell. 14.1-14.40.

1. Hynes, R. O. (1985) Annu. Rev. Cell Biol. 1 , 67-90.

2. Hynes, R. O. (1986) Sci. Ann. 254, 42-51.

6. Kuusela, P. (1978) Nature 276, 718-720.

24. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.,Biol. Chem. 193, 265-275.

13. Lofdahl, S., Guss B., Uhleen, M., Philipson, L. and lindberg, M.(1983) Proc. Natl. Acad. Sci. USA 80, 697-701.

26. Maniatis, T., Fritsch, E. F. and Sambrok, J. (1982). Molecularcloning: a laboratory manual, Cold Spring Harbor Laboratory, New York.

23. March, S. C., Parikh, I. and Cuatrecasas, P. (1974) . Analyt.Biochem. 60, 149-152.

27, Maxam, A. M. and Gilbert, W., (1977), Proc. Natl. Acad. Sci., USA,74, 560.

28. Merrifield, K. B., J. Am. Chem. Soc., 86, pp. 304, (1964)

19. Messing, J. and Carlsson, J. (1984). J. Biotechnol. 1, 253-264.

22. Miekka, S. I., Ingham, K. C. and Menache, D. (1982). Thromb Res. 27,1-14.

15. Nilsson, B., Abrahamsun, l. and Uhlen, M. (1985). EMBO J. 4,1075-1080.

16. Nilsson, B., Moks, T., Jansson, B., Abrahamsen, Elmblad, A.,Holmgren, E., Henrichson, L., Jones, T. A. and Uhlen, M. (1986). ProteinEngineering, In press.

21. Norrander, J., Kempe, T. and Messing, J. (1983). Gene, 26, 101-106.

14. Nossal, N. G. and Heppel, L. A. (1965). J . Biol. Chem. 241,3055-3062.

3. Ruoslahti, E. and Pierschbacher, M. D. (1986). Cell, 44, 517-518.

10. Ryden, C., Rubin, K., Speziale, P., Hook, M., Lindberg, M. andWadstrom, T. (1983), J. Biol. Chem. 258, 3396-3401.

7. Wadstrom, T., Switalski, L., Speziale, P., Rubin, K., Ryden, C.,Froman, G., Faris, A., Lindberg, M., Hook, M., (1985), In Jackson, G. J.(ed), Pathogenesis of Infection. Springer Verlag, Berlin, Heidelberg,New York, Tokyo, pp. 193-207.

4. Woods, A., Couchman, J. R., Johansson, S., and Hook, M. (1986), EMBOJ. 5, 665-670.

5. Yamada, K. M. (1983), Annu. Rev. Biochem. 52, 761-799.

LEGENDS TO THE FIGURES

FIG. 1A. Affinity Chromatography on Fibronectin-Sepharose

A lysate (86 ml) obtained by cold osmotic shock of the E. coli clonepFR001 was mixed with 29 ml of 2M ammonium acetate. The sample was thenapplied to a column (1.9×5.7 cm) equilibrated with 0.5M ammonium acetateat a flow rate of 50 ml/h. After the application of the sample thecolumn was washed with four column volumes of 0.5M ammonium acetate at aflow rate of 20 ml/h. At the same time the sensitivity of the UV-monitorwas increased by a factor of five. The material eluting between 200 and220 ml was pooled, neutralized with ammonium hydroxide and dialysedagainst 10 mM ammonium acetate, pH 7.6.

FIG. 1B. Ion-Exchange Chromatography

A sample (10 ml) of the dialysed material from theaffinity-chromatography described in FIG. 1A was applied to a 1 ml MonoQ (Pharmacia, Sweden) anion column equilibrated with 10 mM ammoniumacetate, pH 7.6. The flow rate was 2 ml/min and the column was eluted bya liner increase in the concentration of ammonium acetate of 25 mM perml. The two peaks (I and II) were pooled as indicated in the figure.

FIG. 2. Polyacrylamide Gel Electrophoresis of Materials From theDifferent Purification Steps of an Osmotic Shock Lysate of E. colipfr001

Material applied (lane 1) to the affinity column(fibronectin-Sepharose), unadsorbed (lane 2) and adsorbed (lane 3)material. Ion exchange chromatography of affinity purified material on aMono Q FPLC column (Pharmacia, Sweden): pool I (lane 4) and pool II(lane 5) as marked in FIG. 1B.

FIG. 3. Restriction Map, Subclones and Deletions of the Insert in pfr001

(A) Restriction map of the 6.5 kb insert. (B) various subclonesconstructed in order to determine the region of the gene which codes forthe fibronectin binding activity. (C) Deletions made from either the 3'or the 5' end of the EcoRI-PstI-fragment by treatment with exonucleaseBal31 (see text for details). The fibronectin binding activity for thediffernt gene products is indicated.

FIG. 4. Polyacrylamide Gel Electrophoresis of the Zz-Fr Protein

Protein A (Sigma Chemical Co, St. Louis; lane A) arid the ZZ-FR fusionprotein (lane B) were reduced and subjected to electrophoresis in SDS ona 5-15% polyacrylamide gel. The gel was subsequently stained withCoomassie blue. In lanes C & D the ZZ-FR fusion protein and protein A,respectively, were fractionated by SDS-electrophoresis on a 5-9%polyacrylamide gel, electroblotted to nitrocellulose paper and probedwith ¹²⁵ I-labelled 29 kD fibronectin fragment as described (Froman etal, 1987). Arrows indicate the migration distance of standard proteinsof known molecular weights.

FIG. 5. Affinity Chromatography of the Zz-Fr Protein

The ZZ-FR fusion protein (0.6 panel A) and protein A (Sigma; ma ChemicalCo; 0,5 mg; panel B) were applied to a 7 ml column of Sepharose 4B CLsubstituted with 29 kD fibronectin fragment. The column was washed with0.5M NaCl in PBS and subsequently eluted with 6M GuHCl in PBS. Fractionsof 2 ml were collected and assayed for protein using the BioRad system.

FIG. 6. Polyacrylamide Gel Electrophoresis of the Zz-Fr Fusion ProteinPreparation, Fractionated by Affinity Chromatography

Material not bound (lane A) and bound and eluted (lane B), respectively,from the 29 kD affinity column was analyzed by SDS-gel electrophoresison 5-15% polyacrylamide gels. The gel was subsequently stained withCoomassie blue. Arrows indicate the migration distance of standardproteins of known molecular weights.

FIG. 7. Inhibition of Binding ¹²⁵ I-Fibronectin to Bacterial Cells.

Staphylococcal cells (5×10⁷) of strain Newman (panel A) or strain 8325-4(panel B) were incubated with 5×10⁴ cpm of ¹²⁵ I-labelled intactfibronectin (o--o) or 29 kD N-terminal fibronectin fragment(.increment.--.increment.) in PBS supplemented with 0,1% BSA, and 0.1%Tween^(R) 80 in a total volume of 0.5 ml for 1 hr in the presence ofincreasing amounts of ZZ-FR fusion protein or protein A (--). Forfurther details of the binding assay used, see Froman et al (1987). Theamounts of ¹²⁵ I-radioactivity associated with bacterial cells wasquantitated and the extent of fibronectin binding calculated. Onehundred percent binding represent ¹²⁵ I-ligand bound to bacteria inabsence of inhibiting protein and 0% binding corresponded toradioactivity recorded from incubation mixtures without bacteria.

FIG. 8A. [SEQ ID NO. 4] Sequence of Nucleotide Coding For FibronectinBinding Protein

The nucleotide sequence coding for the fibronectin binding proteintogether with its corresponding amino acids is given. The different38-amino acids repeats are marked, as well as appearing following almostfour complete 14 amino acids repeats. The restriction sitesBalI-HincII-PvuII have been noted in the figure, as well as have theamino acids sequences corresponding to the different nucleotidesequences.

FIG. 9 The Fibronectin Binding Ability of a Chemically SynthezisedPolypeptide

The fibronectin binding ability of the chemically synthezised 38 aminoacids polypeptide according to Example 2 together with the fibronectinbinding ability of the fragments of said polypeptide have been tested.(*--*) denotes the polypeptide corresponding to the 38(2) repeat of theamino acids sequence; (-) denotes the polypeptide corresponding to aminoacids 1-19 of the amino acids sequence; (o--o) denotes the polypeptidecorresponding to amino acids 20-38 of the amino acids sequence;(.increment.--.increment.) denotes the polypeptide corresponding toamino acids no. 9-30 of the amino acids sequence; and (- - -) thepolypeptide mixture comprising the polypeptidis of amino acids 1-19,amino acids 20-38, amino acids 9-30. The binding ability in percent hasbeen plotted against μg of polypeptide added.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 558 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGCCAAAATAGCGGTAACCAGTCATTCGAGGAAGACACAGAAGAAGACAAACCTAAATAT60                GAACAAGGTGGCAATATCGTAGATATCGATTTTGATAGTGTACCTCAAATTCATGGTCAA120               AATAAAGGTAATCAGTCATTCGAGGAAGATACAGAAAAAGACAAACCTAAGTATGAACAT180               GGCGGTAACATCATTGATATCGACTTCGACAGTGTGCCACATATTCACGGATTCAATAAG240               CACACTGAAATTATTGAAGAAGATACAAATAAAGATAAACCAAGTTATCAATTCGGTGGA300               CACAATAGTGTTGACTTTGAAGAAGATACACTTCCAAAAGTAAGCGGCCAAAATGAAGGT360               CAACAAAGCATTGAAGAAGATACAACACCTCCAATCGTGCCACCAACGCCACCGACACCA420               GAAGTACCAAGTGAGCCGGAAACACCAACGCCACCAACACCAGAAGTACCAAGTGAGCCG480               GAAACACCAACACCACCGACACCAGAAGTGCCGAGTGAGCCAGAAACTCCAACACCGCCA540               ACACCAGAGGTACCAGCT558                                                         (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 342 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GGCCAAAATAGCGGTAACCAGTCATTCGAGGAAGACACAGAAGAAGACAAACCTAAATAT60                GAACAAGGTGGCAATATCGTAGATATCGATTTTGATAGTGTACCTCAAATTCATGGTCAA120               AATAAAGGTAATCAGTCATTCGAGGAAGATACAGAAAAAGACAAACCTAAGTATGAACAT180               GGCGGTAACATCATTGATATCGACTTCGACAGTGTGCCACATATTCACGGATTCAATAAG240               CACACTGAAATTATTGAAGAAGATACAAATAAAGATAAACCAAGTTATCAATTCGGTGGA300               CACAATAGTGTTGACTTTGAAGAAGATACACTTCCAAAAGTA342                                 (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 114 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlyGlnAsnSerGlyAsnGlnSerPheGluGluAspThrGluGluAsp                              151015                                                                        LysProLysTyrGluGlnGlyGlyAsnIleValAspIleAspPheAsp                              202530                                                                        SerValProGlnIleHisGlyGlnAsnLysGlyAsnGlnSerPheGlu                              354045                                                                        GluAspThrGluLysAspLysProLysTyrGluHisGlyGlyAsnIle                              505560                                                                        IleAspIleAspPheAspSerValProHisIleHisGlyPheAsnLys                              65707580                                                                      HisThrGluIleIleGluGluAspThrAsnLysAspLysProSerTyr                              859095                                                                        GlnPheGlyGlyHisAsnSerValAspPheGluGluAspThrLeuPro                              100105110                                                                     LysVal                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3280 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 57..3114                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TGCATTTATTAAGTTTAAAAAATTAATGAATTTTGCATTTAAAGGGAGATATTATA56                    GTGAAAAACAATCTTAGGTACGGCATTAGAAAACATAAATTGGGAGCA104                           ValLysAsnAsnLeuArgTyrGlyIleArgLysHisLysLeuGlyAla                              151015                                                                        GCATCAGTATTCTTAGGAACAATGATCGTTGTTGGGATGGGACAAGAC152                           AlaSerValPheLeuGlyThrMetIleValValGlyMetGlyGlnAsp                              202530                                                                        AAAGAAGCTGCAGCATCAGAACAAAAGACAACTACAGTAGAAGAAAAT200                           LysGluAlaAlaAlaSerGluGlnLysThrThrThrValGluGluAsn                              354045                                                                        GGGAATTCAGCTACTGATAATAAAACAAGTGAAACACAAACAACTGCA248                           GlyAsnSerAlaThrAspAsnLysThrSerGluThrGlnThrThrAla                              505560                                                                        ACTAACGTTAATCATATAGAAGAAACTCGATCATATAACGCAACAGTA296                           ThrAsnValAsnHisIleGluGluThrArgSerTyrAsnAlaThrVal                              65707580                                                                      ACAGAACAACCGTCAAACGCAACACAAGTAACAACTGAAGAAGCACCA344                           ThrGluGlnProSerAsnAlaThrGlnValThrThrGluGluAlaPro                              859095                                                                        AAAGCAGTACAAGCACCACAAACTGCACAACCAGCAAATATAGAAACA392                           LysAlaValGlnAlaProGlnThrAlaGlnProAlaAsnIleGluThr                              100105110                                                                     GTTAAAGAAGAGGTAGTTAAGGAAGAAGCGAAACCTCGAGTTAAGGAA440                           ValLysGluGluValValLysGluGluAlaLysProArgValLysGlu                              115120125                                                                     ACAACACAATCTCAAGACAATAGCGGAGATCAAAGACAAGTAGATTTA488                           ThrThrGlnSerGlnAspAsnSerGlyAspGlnArgGlnValAspLeu                              130135140                                                                     ACACCTAAAAAGGCTACACAAAATCAAGTCGCAGAAACACAAGTTGAA536                           ThrProLysLysAlaThrGlnAsnGlnValAlaGluThrGlnValGlu                              145150155160                                                                  GTGGCACAGCCAAGAACGGCATCAGAAAGTAAGCCAGCTGTGACAAGA584                           ValAlaGlnProArgThrAlaSerGluSerLysProAlaValThrArg                              165170175                                                                     TCAGCAGATGTAGCGGAAGCTAAGGAAGCTAGTAACGCGAAAGTCGAA632                           SerAlaAspValAlaGluAlaLysGluAlaSerAsnAlaLysValGlu                              180185190                                                                     ACGGGTACAGATGTAACAAGTAAAGTTACAGTAGAAATTGGTTCTATT680                           ThrGlyThrAspValThrSerLysValThrValGluIleGlySerIle                              195200205                                                                     GAGGGGCATAACAATACAAATAAAGTAGAACCTCATGCAGGACAACGA728                           GluGlyHisAsnAsnThrAsnLysValGluProHisAlaGlyGlnArg                              210215220                                                                     GCGGTACTAAAATATAAGTTGAAATTTGAGAATGGTTTACATCAAGGT776                           AlaValLeuLysTyrLysLeuLysPheGluAsnGlyLeuHisGlnGly                              225230235240                                                                  GACTACTTTGACTTTACTTTATCAAATAATGTAAATACGCATGGCGTA824                           AspTyrPheAspPheThrLeuSerAsnAsnValAsnThrHisGlyVal                              245250255                                                                     TCAACTGCTAGAAAAGTACCAGAAATTAAAAATGGTTCAGTCGTAATG872                           SerThrAlaArgLysValProGluIleLysAsnGlySerValValMet                              260265270                                                                     GCGACAGGTGAAGTTTTAGAAGGTGGAAAGATTAGATATACATTTACA920                           AlaThrGlyGluValLeuGluGlyGlyLysIleArgTyrThrPheThr                              275280285                                                                     AATGATATTCAAGATAAGGTTGATGTAACGGCTGAACTAGAAATTAAT968                           AsnAspIleGlnAspLysValAspValThrAlaGluLeuGluIleAsn                              290295300                                                                     TTATTTATTGATCCTAAAACTGTACAAACTAATGGAAATCAAACTATA1016                          LeuPheIleAspProLysThrValGlnThrAsnGlyAsnGlnThrIle                              305310315320                                                                  ACTTCAACACTAAATGAAGAACAAACTTCAAAGGAATTAGATGTTAAA1064                          ThrSerThrLeuAsnGluGluGlnThrSerLysGluLeuAspValLys                              325330335                                                                     TATAAAGATGGTATTGGGAATTATTATGCCAATTTAAATGGATCGATT1112                          TyrLysAspGlyIleGlyAsnTyrTyrAlaAsnLeuAsnGlySerIle                              340345350                                                                     GAGACATTTAATAAAGCGAATAATAGATTTTCGCATGTTGCATTTATT1160                          GluThrPheAsnLysAlaAsnAsnArgPheSerHisValAlaPheIle                              355360365                                                                     AAACCTAATAATGGTAAAACGACAAGTGTGACTGTTACTGGAACTTTA1208                          LysProAsnAsnGlyLysThrThrSerValThrValThrGlyThrLeu                              370375380                                                                     ATGAAAGGTAGTAATCAGAATGGAAATCAACCAAAAGTTAGGATATTT1256                          MetLysGlySerAsnGlnAsnGlyAsnGlnProLysValArgIlePhe                              385390395400                                                                  GAATACTTGGGTAATAATGAAGACATAGCGAAGAGTGTATATGCAAAT1304                          GluTyrLeuGlyAsnAsnGluAspIleAlaLysSerValTyrAlaAsn                              405410415                                                                     ACGACAGATACTTCTAAATTTAAAGAAGTCACAAGTAATATGAGTGGG1352                          ThrThrAspThrSerLysPheLysGluValThrSerAsnMetSerGly                              420425430                                                                     AATTTGAATTTACAAAATAATGGAAGCTATTCATTGAATATAGAAAAT1400                          AsnLeuAsnLeuGlnAsnAsnGlySerTyrSerLeuAsnIleGluAsn                              435440445                                                                     CTAGATAAAACTTATGTTGTTCACTATGATGGAGAGTATTTAAATGGT1448                          LeuAspLysThrTyrValValHisTyrAspGlyGluTyrLeuAsnGly                              450455460                                                                     ACTGATGAAGTTGATTTTAGAACACAAATGGTAGGACATCCAGAGCAA1496                          ThrAspGluValAspPheArgThrGlnMetValGlyHisProGluGln                              465470475480                                                                  CTTTATAAGTATTATTATGATAGAGGATATACCTTAACTTGGGATAAT1544                          LeuTyrLysTyrTyrTyrAspArgGlyTyrThrLeuThrTrpAspAsn                              485490495                                                                     GGTTTAGTTTTATACAGTAATAAAGCGAACGGAAATGAGAAAAATGGT1592                          GlyLeuValLeuTyrSerAsnLysAlaAsnGlyAsnGluLysAsnGly                              500505510                                                                     CCGATTATTCAAAATAATAAATTTGAATATAAAGAAGATACAATTAAA1640                          ProIleIleGlnAsnAsnLysPheGluTyrLysGluAspThrIleLys                              515520525                                                                     GAAACTCTTACAGGTCAATATGATAAGAATTTAGTAACTACTGTTGAA1688                          GluThrLeuThrGlyGlnTyrAspLysAsnLeuValThrThrValGlu                              530535540                                                                     GAGGAATATGATTCATCAACTCTTGACATTGATTACCACACAGCTATA1736                          GluGluTyrAspSerSerThrLeuAspIleAspTyrHisThrAlaIle                              545550555560                                                                  GATGGTGGAGGTGGATATGTTGATGGATACATTGAAACAATAGAAGAA1784                          AspGlyGlyGlyGlyTyrValAspGlyTyrIleGluThrIleGluGlu                              565570575                                                                     ACGGATTCATCAGCTATTGATATCGATTACCATACTGCTGTGGATAGC1832                          ThrAspSerSerAlaIleAspIleAspTyrHisThrAlaValAspSer                              580585590                                                                     GAAGCAGGTCACGTTGGAGGATACACTGAGTCCTCTGAGGAATCAAAT1880                          GluAlaGlyHisValGlyGlyTyrThrGluSerSerGluGluSerAsn                              595600605                                                                     CCAATTGACTTTGAAGAATCTACACATGAAAATTCAAAACATCACGCT1928                          ProIleAspPheGluGluSerThrHisGluAsnSerLysHisHisAla                              610615620                                                                     GATGTTGTTGAATATGAAGAAGATACAAACCCAGGTGGTGGTCAGGTT1976                          AspValValGluTyrGluGluAspThrAsnProGlyGlyGlyGlnVal                              625630635640                                                                  ACTACTGAGTCTAATTTAGTTGAATTTGACGAAGAGTCTACAAAAGGT2024                          ThrThrGluSerAsnLeuValGluPheAspGluGluSerThrLysGly                              645650655                                                                     ATTGTAACTGGCGCAGTGAGCGATCATACAACAGTTGAAGATACGAAA2072                          IleValThrGlyAlaValSerAspHisThrThrValGluAspThrLys                              660665670                                                                     GAATATACAACTGAAAGTAATCTGATTGAATTAGTGGATGAATTACCT2120                          GluTyrThrThrGluSerAsnLeuIleGluLeuValAspGluLeuPro                              675680685                                                                     GAAGAGCATGGTCAAGCACAAGGACCAGTCGAGGAAATTACTAAAAAC2168                          GluGluHisGlyGlnAlaGlnGlyProValGluGluIleThrLysAsn                              690695700                                                                     AATCATCATATTTCTCATTCTGGTTTAGGAACTGAAAATGGTCACGGG2216                          AsnHisHisIleSerHisSerGlyLeuGlyThrGluAsnGlyHisGly                              705710715720                                                                  AATTATGACGTGATTGAAGAAATCGAAGAAAATAGCCACGTTGATATT2264                          AsnTyrAspValIleGluGluIleGluGluAsnSerHisValAspIle                              725730735                                                                     AAGAGTGAATTAGGTTATGAAGGTGGCCAAAATAGCGGTAACCAGTCA2312                          LysSerGluLeuGlyTyrGluGlyGlyGlnAsnSerGlyAsnGlnSer                              740745750                                                                     TTCGAGGAAGACACAGAAGAAGACAAACCTAAATATGAACAAGGTGGC2360                          PheGluGluAspThrGluGluAspLysProLysTyrGluGlnGlyGly                              755760765                                                                     AATATCGTAGATATCGATTTTGATAGTGTACCTCAAATTCATGGTCAA2408                          AsnIleValAspIleAspPheAspSerValProGlnIleHisGlyGln                              770775780                                                                     AATAAAGGTAATCAGTCATTCGAGGAAGATACAGAAAAAGACAAACCT2456                          AsnLysGlyAsnGlnSerPheGluGluAspThrGluLysAspLysPro                              785790795800                                                                  AAGTATGAACATGGCGGTAACATCATTGATATCGACTTCGACAGTGTG2504                          LysTyrGluHisGlyGlyAsnIleIleAspIleAspPheAspSerVal                              805810815                                                                     CCACATATTCACGGATTCAATAAGCACACTGAAATTATTGAAGAAGAT2552                          ProHisIleHisGlyPheAsnLysHisThrGluIleIleGluGluAsp                              820825830                                                                     ACAAATAAAGATAAACCAAGTTATCAATTCGGTGGACACAATAGTGTT2600                          ThrAsnLysAspLysProSerTyrGlnPheGlyGlyHisAsnSerVal                              835840845                                                                     GACTTTGAAGAAGATACACTTCCAAAAGTAAGCGGCCAAAATGAAGGT2648                          AspPheGluGluAspThrLeuProLysValSerGlyGlnAsnGluGly                              850855860                                                                     CAACAAACGATTGAAGAAGATACAACACCTCCAATCGTGCCACCAACG2696                          GlnGlnThrIleGluGluAspThrThrProProIleValProProThr                              865870875880                                                                  CCACCGACACCAGAAGTACCAAGTGAGCCGGAAACACCAACGCCACCA2744                          ProProThrProGluValProSerGluProGluThrProThrProPro                              885890895                                                                     ACACCAGAAGTACCAAGTGAGCCGGAAACACCAACACCACCGACACCA2792                          ThrProGluValProSerGluProGluThrProThrProProThrPro                              900905910                                                                     GAAGTGCCGAGTGAGCCAGAAACTCCAACACCGCCAACACCAGAGGTA2840                          GluValProSerGluProGluThrProThrProProThrProGluVal                              915920925                                                                     CCAGCTGAACCTGGTAAACCAGTACCACCTGCCAAAGAAGAACCTAAA2888                          ProAlaGluProGlyLysProValProProAlaLysGluGluProLys                              930935940                                                                     AAGCCTTCTAAACCAGTGGAACAAGGTAAAGTAGTAACACCTGTTATT2936                          LysProSerLysProValGluGlnGlyLysValValThrProValIle                              945950955960                                                                  GAAATCAATGAAAAGGTTAAAGCAGTGGCACCAACTAAAAAACCACAA2984                          GluIleAsnGluLysValLysAlaValAlaProThrLysLysProGln                              965970975                                                                     TCTAAGAAATCTGAACTACCTGAAACAGGTGGAGAAGAATCAACAAAC3032                          SerLysLysSerGluLeuProGluThrGlyGlyGluGluSerThrAsn                              980985990                                                                     AAAGGTATGTTGTTCGGCGGATTATTCAGCATTCTAGGTTTAGCATTA3080                          LysGlyMetLeuPheGlyGlyLeuPheSerIleLeuGlyLeuAlaLeu                              99510001005                                                                   TTACGCAGAAATAAAAAGAATCACAAAGCATAATAAACAAAAAT3124                              LeuArgArgAsnLysLysAsnHisLysAla*                                               10101015                                                                      TGACGGGTTTATTTCATAAATTATATGAAGTAAGCCTGTTTTTTTATATTAAATCAAATT3184              TCTAATAGAAAATTAGAGTGTTTTCTGATTGCTTCATTGGTTTATGTCTGATGATTGATA3244              ACGAACTGAGAGATAAAGTTAGAATTTTAAAGTAGT3280                                      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We claim:
 1. A vaccine composition comprising an effective amount of aprotein encoded by a hybrid DNA molecule comprising a nucleotidesequence from Staphylococcus aureus encoding an isolated protein havingfibronectin binding activity, whereby said protein induces immunizationby forming antibodies in a mammal against Staphylococcus aureus toprovide protection for clinical symptoms to Staphylococcus aureuswherein the hybrid DNA molecule comprises one or more of the nucleotidesequences selected from the group consisting of nucleotides 1-114 of[SEQ ID NO. 2], nucleotides 115-228 of [SEQ ID NO. 2], and nucleotides229-342 of [SEQ ID NO. 2]: and a pharmaceutical acceptable vaccinecarrier therefor.
 2. A vaccine composition comprising an effectiveamount of a protein encoded by a hybrid DNA molecule comprising anucleotide sequence from Staphylococcus aureus encoding an isolatedprotein having fibronectin binding activity, whereby said proteininduces immunization by forming antibodies in a mammal againstStaphylococcus aureus to provide protection for clinical symptoms toStaphylococcus aureus, wherein the hybrid DNA molecule comprises thenucleotide sequence set forth in [SEQ ID NO. 4] and a pharmaceuticallyacceptable vaccine carrier therefor.
 3. The vaccine composition of claim2, wherein said mammal is a ruminant.
 4. The vaccine composition ofclaim 2, wherein said mammal is a bovine.
 5. The vaccine composition ofclaim 2, wherein said mammal is a human.
 6. A vaccine compositioncomprising an effective amount of a protein encoded by a plasmid orphage comprising a nucleotide sequence from Staphylococcus aureusencoding an isolated protein having fibronectin binding activity,whereby said sequence of the plasmid or phage comprises [SEQ ID NO. 4]and a pharmaceutically acceptable vaccine carrier therefor.
 7. A vaccinecomposition comprising an effective amount of a protein, whereby saidprotein induces immunization by forming antibodies againstStaphylococcus aureus to provide protection for clinical symptoms toStaphylococcus aureus, wherein the protein comprises the amino acidsequence of [SEQ ID NO. 3] and a pharmaceutically acceptable vaccinecarrier therefor.
 8. A vaccine composition comprising an effectiveamount of a protein encoded by plasmid pFR001 as contained inEscherichia coli strain 259 having the deposit number DSM 4124, saidplasmid comprising a nucleotide sequence from Staphylococcus aureusencoding an isolated protein having fibronectin binding activity,whereby said protein induces immunization in a mammal by formingantibodies against Staphylococcus aureus, and a pharmaceuticallyacceptable vaccine carrier therefor.